Creation of a fully functional human chimeric DNA repair protein. Combining O6-methylguanine dna methyltransferase (MGMT) and AP endonuclease (APE/Redox effector factor 1 (Ref 1) DNA repair proteins

Warren K. Hansen, Walter A. Deutsch, Adley Yacoub, Yi Xu, David A. Williams, Mark Kelley

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

A dose-limiting toxicity of certain chemotherapeutic alkylating agents is their toxic effects on nontarget tissues such as the bone marrow. To overcome the myelosuppression observed by chemotherapeutic alkylating agents, one approach is to increase the level of DNA repair proteins in hematopoietic stem and progenitor cells. Toward this goal, we have constructed a human fusion protein consisting of O6-methylguanine DNA methyltransferase coupled with an apurinic endonuclease, resulting in a fully functional protein for both O6-methylguanine and apurinic/apyrimidinic (AP) site repair as determined by biochemical analysis. The chimeric protein protected AP endonuclease-deficient Escherichia coli cells against methyl methanesulfonate and hydrogen peroxide (H2O2) damage. A retroviral construct expressing the chimeric protein also protected HeLa cells against 1,3-bis(2-chloroethyl)-1- nitrosourea and methyl methanesulfonate cytotoxicity either when these agents were used separately or in combination. Moreover, as predicted from previous analysis, truncating the amino 150 amino acids of the apurinic endonuclease portion of the O6-methylguanine DNA methyl-transferase-apurinic endonuclease protein resulted in the retention of O6-methylguanine DNA methyltransferase activity but loss of all AP endonuclease activity. These results demonstrate that the fusion of O6-methyl-guanine DNA methyltransferase and apurinic endonuclease proteins into a combined single repair protein can result in a fully functional protein retaining the repair activities of the individual repair proteins. These and other related constructs may be useful for protection of sensitive tissues and, therefore, are candidate constructs to be tested in preclinicai models of chemotherapy toxicity.

Original languageEnglish
Pages (from-to)756-762
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number2
DOIs
StatePublished - Jan 9 1998

Fingerprint

Endonucleases
Methyltransferases
DNA Repair
Oxidation-Reduction
Repair
DNA-(Apurinic or Apyrimidinic Site) Lyase
DNA
Proteins
Methyl Methanesulfonate
Alkylating Agents
Hematopoietic Stem Cells
Toxicity
Fusion reactions
O-(6)-methylguanine
Tissue
Carmustine
Chemotherapy
Poisons
Guanine
Cytotoxicity

ASJC Scopus subject areas

  • Biochemistry

Cite this

Creation of a fully functional human chimeric DNA repair protein. Combining O6-methylguanine dna methyltransferase (MGMT) and AP endonuclease (APE/Redox effector factor 1 (Ref 1) DNA repair proteins. / Hansen, Warren K.; Deutsch, Walter A.; Yacoub, Adley; Xu, Yi; Williams, David A.; Kelley, Mark.

In: Journal of Biological Chemistry, Vol. 273, No. 2, 09.01.1998, p. 756-762.

Research output: Contribution to journalArticle

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